The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

Khomaini Hasan, Artur Gora, Jan Brezovsky, Radka Chaloupkova, Hana Moskalikova, Andrea Fortova, Yuji Nagata, Jiri Damborsky, Zbynek Prokop

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K0.5 for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 possesses a unique halide-stabilising tyrosine residue, Y109, in place of the conventional tryptophan. This is the first natural haloalkane dehalogenase that stabilises its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

    Original languageEnglish
    Pages (from-to)3149-3159
    Number of pages11
    JournalFEBS Journal
    Volume280
    Issue number13
    DOIs
    Publication statusPublished - 2013 Jul

    Keywords

    • catalytic mechanism
    • enzyme kinetics
    • halide-stabilizing residue
    • haloalkane dehalogenase
    • substrate specificity

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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  • Cite this

    Hasan, K., Gora, A., Brezovsky, J., Chaloupkova, R., Moskalikova, H., Fortova, A., Nagata, Y., Damborsky, J., & Prokop, Z. (2013). The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58. FEBS Journal, 280(13), 3149-3159. https://doi.org/10.1111/febs.12238